Project Summary Cancer metastasis accounts for over 90% of all cancer deaths. A limiting step in cancer metastatic cascade is for tumor cells to migrate towards, interact with and squeeze through the blood vessel wall before disseminating to secondary tumor sites via the blood circulation. Biophysical forces, including interstitial and intramural flows, have shown to play critical roles in regulating adhesion molecules, spatial cytokine distributions and tissue architecture; all of which contribute to tumor cell invasion within 3D biomatrix. Despite the clinical importance, roles of biophysical forces in tumor cell transendothelial migration (TEM) are poorly understood. This is in part due to the lack of in vitro tools that are able to follow tumor cell transmigration events in real time, and with well controlled biological flows. Current animal cell invasion assay, the Boyden Chamber, is limited because it is difficult to recreate complex tumor microenvironment. In addition, the results are population based at two time end points. Intravital imaging has advanced significantly our understanding about the interplays between tumor microenvironment and TEM in a physiologically realistic setting. However, it is difficult to dissect the contribution of individual environmental cues to TEM processes. The goals of the proposed research are to develop a physiologically realistic microfluidic model with well controlled tumor microenvironment for studies of tumor cell TEM processes; and to identify tumor microenvironment that promotes TEM. To achieve these goals, we will develop an organotypic microfluidic model for real time imaging of tumor cell TEM events under well controlled micro-environment. We will use the location of spheroid and cell streaming event to guide TEM imaging sites. Using the microfluidic model, we will explore the relations between single tumor cell properties and TEM activities under well controlled interstitial and intramural flows. Previous work from the PI?s lab and others have indicated that interstitial flows critically regulate tumor cell migration within 3D biomatrix. Here, we hypothesize that tumor cells? TEM capabilities are closed correlated with cells? microenvironment including fluid flows. The proposed project is innovative because it represents the first generation of organotypic microfluidic platform that includes both interstitial and intramural flows, moving the current microfluidic tumor model towards a physiologically realistic direction. Lessons learned here will eventually lead to knowledge important for developing novel diagnostic or/and treatment strategies for cancer. This platform can be readily extended for use in other biological systems where TEMs are important including immune cell trafficking.